Yeast strains, such as Pichia pastoris, are commonly used for the production of heterologous proteins. P. pastoris has become a popular model system for the study of peroxisome biogenesis (Gould et al., Yeast 8:613-628 (1992)), autophagy (Tuttle and Dunn, J. Cell Sci. 108:25-35 (1995); Sakai et al., J. Cell Biol. 141:625-636 (1998)) and the organization and biogenesis of the organelles of the secretory pathway (Rossanese et al., J. Cell Biol. 145:69-81 (1999)). The development of simple DNA transformation systems, (see Cregg et al., Mol. Cell. Biol. 5:3376-3385 (1985)) and the availability of selectable marker genes have been of great importance in conducting the above experiments. Currently, the biosynthetic marker genes ADE1, ARG4, HIS4 and URA3 are used in conjunction with the corresponding auxotrophic host strains to select for transformed cells. See Lin Cereghino et al., Gene 263:159-169 (2001). The use of dominant selectable markers to identify transformants is also possible, but markers are limited to the Sh ble gene from Streptoalloteichus hindustanus, which confers resistance to the drug Zeocin (Higgins et al., Methods Mol. Biol. 103:41-53 (1998)), and the blasticidin S deaminase gene from Aspergillus terreus, which confers resistance to the drug blasticidin (Kimura et al., Mol. Gen. Genet. 242:121-129 (1994)).
Stable integration of cloned DNA segments into the yeast genome through homologous recombination is well known in the art. See e.g., Orr-Weaver et al., Proc. Natl. Acad. Sci. USA 78:6364-6358 (1981). More recently, methods have been developed in S. cerevisiae to generate yeast strains containing DNA integrated at multiple unlinked sites by homologous recombination using molecular constructs containing the URA3 marker genes. See e.g., Alani et al., Genetics 116: 541-545 (1987). In Pichia pastoris, yeast strains have been developed containing integrated DNA using constructs encoding the URA5 marker. See e.g., Nett and Gerngross, Yeast 20: 1279-1290 (2003).
Both the PpURA3 and the PpURA5 genes can be used repeatedly after counterselection on medium containing 5-fluoroorotic acid (5FOA). However, unlike ura auxotrophic strains in Saccharomyces cerevisiae (S.c.), the P. pastoris ura3 and ura5 auxotrophs have significantly reduced growth rates which are likely due to an inability of P. pastoris to take up uracil from the medium. See Lin Cereghino et al., Gene 263:159-169 (2001). These plasmid gene cassettes can potentially be reused indefinitely, however, another disadvantage of these as recyclable markers is the additional time required to recover the marker, thereby at least doubling the time necessary for one round of genetic modification.
Extensive genetic engineering projects, requiring the expression of several genes in parallel, necessitate the availability of counterselectable markers and plasmids for stable genetic integration of heterologous proteins into the host genome. Presently available auxotrophic strains of P. pastoris suffer the disadvantage of gene reversion. A high reversion rate decreases the usefulness of auxotrophic strains because revertant colonies are misidentified as false-positive transformants.
What is needed, therefore, is a method for stable genetic introduction of several heterologous genes into the genome of Pichia pastoris without the need for recyclable or multiple auxotrophic markers.